Bioleaching of Spent Fluid Catalytic Cracking Catalyst Using Aspergillus niger

Abstract

The use of the fungus Aspergillus niger for the bioleaching of heavy metals from spent catalyst was investigated, with fluid catalytic cracking (FCC) catalyst as a model. Bioleaching was examined in batch cultures with the spent catalysts at various pulp densities (1-12%). Chemical leaching was also performed using mineral acids (sulphuric and nitric acids) and organic acids (citric, oxalic and gluconic acids), as well as a mixture of organic acids at the same concentrations as that biogenically produced. It was shown that bioleaching realised higher metal extraction than chemical leaching, with A. niger mobilizing Ni (9%), Fe (23%), Al (30%), V (36%) and Sb (64%) at 1% pulp density. Extraction efficiency generally decreased with increased pulp density. Compared with abiotic controls, bioleaching gave rise to higher metal extractions than leaching using fresh medium and cell-free spent medium. pH decreased during bioleaching, but remained relatively constant in both leaching using fresh medium and cell-free spent medium, thus indicating that the fungus played a role in effecting metal extraction from the spent catalyst.

Full text

Journal of Biotechnology 116 (2005) 159–170
Bioleaching of spent fluid catalytic cracking catalyst
using Aspergillus niger
Khin Moh Moh Aung, Yen-Peng Ting∗
Department of Chemical and Biomolecular Engineering, National University of Singapore,
10 Kent Ridge Crescent, Singapore 119260, Singapore
Received 3 September 2003; received in revised form 29 July 2004; accepted 18 October 2004
Abstract
The use of the fungus Aspergillus niger for the bioleaching of heavy metals from spent catalyst was investigated, with fluid
catalytic cracking (FCC) catalyst as a model. Bioleaching was examined in batch cultures with the spent catalysts at various pulp
densities (1–12%). Chemical leaching was also performed using mineral acids (sulphuric and nitric acids) and organic acids
(citric, oxalic and gluconic acids), as well as a mixture of organic acids at the same concentrations as that biogenically produced.
It was shown that bioleaching realised higher metal extraction than chemical leaching, with A. niger mobilizing Ni (9%), Fe
(23%), Al (30%), V (36%) and Sb (64%) at 1% pulp density. Extraction efficiency generally decreased with increased pulp
density. Compared with abiotic controls, bioleaching gave rise to higher metal extractions than leaching using fresh medium and
cell-free spent medium. pH decreased during bioleaching, but remained relatively constant in both leaching using fresh medium
and cell-free spent medium, thus indicating that the fungus played a role in effecting metal extraction from the spent catalyst.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Bioleaching; Aspergillus niger; Solid wastes; Spent catalyst; Two-step bioleaching
1. Introduction
Bioleaching may be described as an interaction be-
tween metals and microorganisms that causes the sol-
ubilization of the metals, and is based on the ability
of microorganisms to transform solid compounds, thus
resultinginsolubleandextractableelementswhichcan
∗Correspondingauthor.Tel.:+65 68742190;fax: +65 67791936.
E-mail address: chetyp@nus.edu.sg (Y.-P. Ting).
be recovered (Brandl et al., 1997). The ability of a va-
riety of microorganisms to mobilize and leach met-
als from solid materials is based on three principles,
namely (i) the transformation of organic or inorganic
acids (protons); (ii) oxidation and reduction reactions
and (iii) the excretion of complexing agents. Metals
can be leached either directly (i.e. physical contact be-
tween microorganisms and solid material) or indirectly
(e.g.bacterial oxidation ofFe2+ toFe3+ which catalyses
metal solubilization as an electron carrier) (Brandl et
0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2004.10.008

160 K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170
al., 1997). Microbial leaching technologies have been
used on an industrial scale for the recovery of cop-
per, gold, uranium and zinc from low-grade ores, or
from low-grade mineral resources (Brombacher et al.,
1997).
Three groups of microorganisms are used in leach-
ing: autotrophic bacteria, heterotrophic bacteria and
fungi. The use of autotrophic Thiobacilli is advanta-
geous because no organic carbon source is needed for
their growth. On the other hand, heterotrophic bacteria
andfungicanbeused with higher pHs (i.e. alkaline and
acid-consuming materials) (Schinner and Burgstaller,
1989). The most effective and common bacteria for
metal solubilization belong to the genus Bacillus of
which the chemolithoautotrophs Thiobacillus ferroox-
idans and T. thiooxidans are of industrial importance.
In addition, the Aspergillus and Penicillium genera are
themostwellstudiedfungiusedinbioleachingstudies.
In many chemical industries, large quantities of
solid catalysts are routinely used. These catalysts often
require replacement after 2 or 3 years of operation. For
instance, in fluid catalytic cracking (FCC), which is a
major secondary conversion process in petroleum re-
fining,about 4 ×108kg ofspentcatalysts are generated
annually on a global scale (Furimsky, 1996).
Currently, spent catalysts are managed industrially
via(i) chemicalrecoveryandrecyclingof valuablemet-
als for different applications, (ii) regeneration (to ex-
tend their operational life) for reuse and (iii) landfilling
(for ultimate disposal). The recovery of valuable met-
als, while conferring economic advantage, entails the
use of acids in large scale processing operations, which
generatelargevolumes of potentially hazardous wastes
and gaseous emissions. The regeneration of spent cata-
lysts,unfortunately,can only beappliedfor a fewtimes,
and on a limited number of catalytic systems. Landfill-
ing as the means for disposal of spent catalysts is be-
coming increasingly difficult, due both to decreasing
availability of landfill space as well as the concern for
pollution arising from possible leaching of heavy met-
als. Thus, the high cost and the negative environmental
impacts of conventional methods warrant investigation
into the possible use of bioleaching technology as an
alternative in the extraction of metals from solid waste
materials.
Heavy metals may be present as part of the original
catalyst (for example, cobalt or nickel in hydroprocess-
ingcatalysts) ormay beaccumulatedduring use(forex-
ample, nickel or vanadium in FCC catalysts). In effect,
these waste materials containing high metal concentra-
tions may be considered as ‘artificial ores’, as they can
serve as secondary raw materials with the consequent
reductioninthedemand for primary mineral resources.
In addition, the removal of metals from these industrial
wastes brings about a detoxification of the residues
and thus improves the environment (Brombacher et al.,
1997; Brandl, 2001).
Currently, the predominant use of spent FCC cat-
alyst is in the area of construction materials such
as the filler for asphalt as well as the production of
bricks and cement (Furimsky, 1996). With bioleach-
ing, the leached and recovered metals may be re-
cycled and re-used as raw materials in the metal-
manufacturing industries. Using spent FCC catalyst as
amodel,the objective ofthisworkis to demonstrate the
use of Aspergillus niger in leaching metals from spent
catalyst.
2. Materials and methods
2.1. Fungal strain and growth conditions
A. niger was obtained from Dr. H. Brandl (Univer-
sity of Zurich, Switzerland). The fungus was cultivated
on 3.9% (w/v) potato dextrose agar (Becton Dickin-
son, USA) plates and was kept in an incubator for 7
days at 30◦C. Sterile deionised water was used to re-
cover the spores, which were counted under an optical
microscope (Olympus CX 40) at 400 times magnifi-
cation using a haemacytometer. The spore suspension
wasthendilutedwithdeionisedwaterand standardized
to 1×107spores ml−1of spore suspension.
To culture in liquid medium, 1ml of spore suspen-
sionwasadded to 100 ml of sucrose medium with com-
position 100gl−1sucrose, 1.5 gl−1NaNO3,0.5gl−1
KH2PO4, 0.025gl−1MgSO4·7H2O, 0.025 g l−1KCl
and 1.6 gl−1yeast extract (Bosshard et al., 1996). The
cultures, in 250 ml Erlenmeyer flasks, were maintained
at 30◦C in a water bath at 120rpm.
2.2. Catalyst (silica-alumina (zeolitic) catalyst)
Fresh and spent catalysts used in this project
were kindly provided by Singapore Refining Com-
pany (SRC). The catalyst is silica-alumina based and

K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170 161
is commonly used in the Fluid Catalytic Cracking
unit (hence designated FCC catalyst) in petroleum
refining.
2.3. Characterization of the catalyst
2.3.1. Catalyst composition
The elemental composition of the fresh, spent
and bioleached spent catalyst was determined using
three different methods: chemical analysis, scanning
electron microscope-energy dispersive X-ray analysis
(SEM-EDX) and CHN analysis.
2.3.1.1. Chemical analysis. Metal analysis was per-
formed using inductively coupled plasma optical emis-
sion spectrometer (ICP-OES, Perkin-Elmer, Optima
3000V) after acid digestion of the samples. Approx-
imately, 0.2g of spent catalyst was heated in a furnace
at 500 ◦C for 4 h before it was placed in a Teflon bottle.
Five-milliliterhydrofluoric acid (48%), 4 ml perchloric
acid (70–72%) and 10ml 1:1 HCl were added to the
bottle and placed in a heated water bath (80–90◦C).
Heating continued until white fumes appeared. After
cooling, 2ml H2O2(30%) was added into the bottle
and the heating resumed. The solution was concen-
trated to about 2ml. The digestate was cooled, trans-
ferred to 25ml volumetric flask and diluted to volume
using 20% HCl and analysed after filtration through
0.45␮m glass fibre filter.
2.3.1.2. SEM-EDX. The fresh, spent and bioleached
spent catalysts were subjected to semi quantitative
analysis using a JEOL JSM-5600LV scanning elec-
tron microscope-energy dispersive X-ray analysis. The
samples were sputter-coated with platinum (JFC-1300,
Jeol, Tokyo) after spreading the sample on metallic
studs with carbon tape prior to observation under the
electron microscope.
2.3.1.3. CHN analysis. Carbon content of the fresh,
spent and bioleached spent catalysts were determined
using a CHNS/O analyser (Perkin-Elmer 2400 Series
II). One to two milligrams of the catalyst samples were
weighed into small tin vials with Perkin-Elmer AD-6
Ultramicrobalance. Four standard vials were also pre-
pared using acetanilide (Elemental Microanalysis Ltd.,
UK) containing 71.09% C, 6.71% H, 10.36% N and
11.84% O. The vials were placed in the auto-sampler
installedonthe analyser.Beforethesampleswere anal-
ysed, a series of blank runs (using empty vials) fol-
lowed by three standard runs were carried out. These
vialswerecombusted under oxygen stream in a furnace
(975◦C).
2.3.2. Particle size distribution, surface area and
morphology of catalysts
The particle size distribution was determined using
aCoulter LS 230 particle sizeanalyserwith a size range
0.04–2000␮m.
The specific surface area, total pore volume and
the average pore diameter of the fresh, spent and bi-
oleached catalysts were determined using a high speed
gas adsorption analyser (Nova 3000 Version 6.07,
Quantachrome Corporation) with Brunauer–Emmett–
Teller (BET) method.
The Jeol JSM-5600 LV Scanning Electron Micro-
scope was used to observe the morphology of the cata-
lysts. Samples were spread on metallic studs using car-
bon tape and coated with platinum. Image analysis was
conducted under an accelerating voltage of 15–20kV,
and under high vacuum.
2.3.3. Toxicity characteristic leaching procedure
(TCLP) tests
Toxicity characteristic leaching procedure tests
were performed on the catalysts according to
U.S. Environmental Protection Agency (EPA) SW
846 method 1311 (Toxicity Characteristic Leaching
Procedure, 1998). Using TCLP extraction fluid # 1
(pH 4.93±0.05) for the leaching test, the extract was
analysed immediately using ICP-OES after filtering
through a 0.45␮m glass fibre filter.
2.4. Chemical leaching
Chemical leaching tests were carried out with ox-
alic, citric, gluconic, nitric and sulphuric acids (20, 50
and 100mM) as well as a mixture of commercial cit-
ric, oxalic and gluconic acids at concentrations equal
to the acids produced in the bioleaching processes. The
leaching tests were conducted using 100ml of acid in
250 ml Erlenmeyer flask with 2% (w/v) pulp density of
spent catalyst at 30◦C and 120rpm. All experiments
were conducted in duplicates. Samples were collected,
filtered and analysed for metals using ICP-OES.

162 K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170
2.5. Bioleaching of spent catalyst by A.niger
Bioleaching was performed in 250ml Erlenmeyer
flasks with 100ml of sucrose medium with the spent
catalyst at various pulp densities (1, 2, 4, 8 and 12%
w/v). One-step bioleaching (where the fungus was in-
cubated together with the medium and catalyst) and
two-step bioleaching (where the fungus was first cul-
tured in sucrose medium without catalyst for two days,
after which sterilised catalyst was added) were inves-
tigated. In spent medium leaching, the fungus was
first cultured in sucrose medium for 14 days. The
culture was passed through glass wool followed by
centrifugation and membrane filtration (1.5, 0.45 and
finally 0.2␮m) (Whatman®) to obtain the cell-free
spent medium before sterilised catalyst was added to
the filtrate. Control experiments were conducted us-
ing deionised water and fresh sucrose medium. Ster-
ile experimental set-up was achieved by autoclaving at
121◦C for 15min prior to inoculation.
The cultures were incubated at 30◦C with rotary
shaking at 120rpm. Samples (5ml) were withdrawn
at regular intervals for the following analyses: (i)
pH, (ii) heavy metal concentrations, (iii) sugar con-
centrations and (iv) organic acid concentrations. The
biomassdry weight was alsodeterminedatregular time
intervals.
2.6. Analytical methods
2.6.1. Analysis of sugars and organic acids
Sucrose was provided as the sole carbon source
for A. niger and was hydrolysed to glucose and fruc-
tose. The concentration of glucose was measured us-
ing YSI biochemistry analyser with a glucose sensor.
Concentration of sucrose and fructose, as well as the
biogenically produced organic acids (citric, gluconic
and oxalic) were determined using high performance
liquid chromatography (HPLC) with variable wave-
length detector (VWD) at 210 nm for the organic acids
and refractive index detector (RID) for the sugars. An
Aminex HPX-87H, 300mm ×7.8 mm (BioRad) col-
umn with microguard cation H precolumn (BioRad)
was used. The operation was carried out at 30◦C, with
maximum operation pressure at 1×107Pa. The mo-
bile phase used was 0.005M sulphuric acid at a flow
rateof 0.5 mlmin−1.Prior toanalysis,the sampleswere
filtered with 0.2 ␮m nylon membrane syringe filter (Ti-
tan) to protect the column from being clogged by fine
particles in the samples.
2.6.2. Analysis of metal composition
Metal analysis was performed using inductively
coupled plasma optical emission spectrometer (ICP-
OES, Perkin-Elmer, Optima 3000V) at the follow-
ing wavelengths (nm): Al (308.215), Fe (259.940),
Ni (231.604), Sb (206.833), V (292.402). ICP multi-
element standard IV (Merck) at 1000mgl−1was used
to prepare the calibration standards after dilution with
0.1M nitric acid. Additional single element standards
for Sb and V were also prepared. Samples from bi-
oleaching and chemical leaching were filtered through
a Whatman Autovial®(0.45␮m) before analysis.
2.6.3. Determination of biomass
To determine the fungal biomass (in the absence
of the spent catalyst in the medium), the culture broth
was filtered through a Buchner funnel, after which the
biomass was washed with deionised water and trans-
ferred to pre-weighed evaporating dish and dried at
80◦C for 24h, cooled in a desiccator and weighed.
In experiments where spent catalyst was present in
the medium, the culture broth was filtered through a
Bunchner funnel and the biomass was washed with
deionised water until the filtrate was clear of the spent
catalyst. The biomass together with any residual spent
catalyst was dried at 80◦C for 24 h, cooled in a des-
iccator and weighed, and finally ashed at 500◦C for
4 h. The weight of the biomass was calculated from the
difference between these two temperature settings.
3. Results and discussion
3.1. Characterization of the catalyst
3.1.1. Catalyst composition
From Table 1, it can be seen that nickel and vana-
dium were not found in the fresh catalyst, but in the
spent and bioleached spent catalyst. These metals must
have been accumulated during use; these contaminant
metalsoriginatedfromthehigh-molecularweightfrac-
tion of the feed to the FCC. Sun et al. (1998) also re-
ported a similar amount of vanadium and nickel on
spent FCC catalyst. The presence of antimony in the
spent catalyst is due to injection of the metal into the

K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170 163
Table 1
Elemental compositions of fresh (FFCC), spent (SFCC) and bioleached spent FCC (BSFCC) catalysts
Elements Elemental composition of catalysts (wt.%)
FFCC SFCC BSFCC
Ala19.09 ±2.83 17.5 ±2.49 (33.08 as Al2O3) 9.38 ±0.62
Fea0.28 ±0.10 0.56 ±0.12 0.37 ±0.02
Niand 0.26 ±0.07 0.24 ±0.01
Sband 0.03 ±0.005 0.026 ±0.001
Vand 0.39 ±0.07 0.22 ±0.01
Ob77.53 ±2.10 54.44 ±0.61 82.72 ±4.84
Sb0.47 ±0.08 nd nd
Sib10.48 ±0.93 22.46 ±0.77 9.19 ±2.95
Cc0.15 ±0.03 0.44 ±0.03 1.66 ±0.01
nd: not detected.
aAnalysed using ICP-OES.
bAnalysed using SEM-EDX.
cAnalysed using CHN analyser.
fresh feed to passivate nickel compounds in the FCC
feed (Sadeghbeigi, 1995). The semi quantitative SEM-
EDX results showed that the most abundant element in
all the catalysts was oxygen; the high concentration of
oxygen was due to the presence of metals as metal ox-
ides.Notunexpectedly,the carbon content of the SFCC
was higher than that of FFCC, indicating that some
residual coke still remained in the spent catalyst af-
ter regeneration. (The low amount in the spent catalyst
may be attributed to its withdrawal downstream of the
regeneratorintheFCC unit;the FCC unitincorporates a
catalyst regenerator immediately after the reactor, and
the regenerated catalyst is recycled back to the reac-
tor.) The much higher carbon content of the BSFCC
may be attributed to the deposition of some residual
carbon from the sugars in the culture medium. (It was
confirmed when washing the BSFCC with deionised
water and hot water resulted in a carbon content of 3.4
(±0.02) and 1.66 (±0.01)wt.%, respectively.) From
the analyses, it is evident that the spent catalyst con-
tained the heavy metals, nickel, antimony and vana-
dium.
3.1.2. Particle size distribution, surface area and
morphology of catalysts
3.1.2.1. Particle size distribution. The mean particle
diameter of the fresh, spent and bioleached FCC cat-
alysts were 70.6, 93.7 and 81.1␮m, respectively. The
higher mean particle diameter of spent catalyst is due
to the deposition of coke and other metal contaminants
during the reaction. Bioleaching resulted in approxi-
mately 13% reduction in the mean particle diameter;
the deposited coke and some metal components were
leached out during bioleaching.
Particle size distribution analysis showed that the
particle size of the catalyst was less than 200 ␮minall
cases, and particles smaller than 150 ␮m accounted for
about 90% of the total volume. The particle size distri-
bution of the fresh catalyst was consistent with that of
a typical fresh FCC catalyst (Sadeghbeigi, 1995). The
volume percentage of fresh catalyst with particle size
less than 40␮m was significantly higher than that of
spent and bioleached catalysts, possibly because parti-
cles in this size range escaped the unit via the cyclones
after a few cycles.
3.1.2.2. BET analysis. Results of the BET analysis
for the fresh catalyst (Table 2) is consistent with the
Table 2
Specific surface area, total pore volume and average pore diameter
of fresh (FFCC), spent (SFCC) and bioleached spent FCC (BSFCC)
catalysts
FFCC SFCC BSFCC
Specific
surface area
(m2g−1)
121.55 ±10.34 83.31 ±0.68 133.47 ±3.35
Total pore
volume
(cm3g−1)
0.14 ±0.006 0.133 ±0.01 0.188 ±0.003
Average pore
diameter
(˚
A)
46.83 ±2.77 63.85 ±2.97 56.40 ±0.72

164 K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170
literature (Wojciechowski and Corma, 1986). BET
analysisalso showedthat the specificsurfacearea ofthe
spentcatalyst was lowerthan the fresh catalystby about
31.5%, possibly due to the fouling of contaminant met-
als. Interestingly, bioleaching of the spent catalyst in-
creased its specific surface area beyond the original
fresh catalyst by about 10%.
3.1.2.3. Determination of surface morphology by
SEM. The SEM photomicrograph of the fresh FCC
(Fig. 1a) revealed the spherical shape of the catalysts,
with considerable variation in particle size. In the case
of the spent FCC (Fig. 1b), however, a more homoge-
neousdistribution ofthe discrete particles,withno fines
was seen. These figures are consistent with the parti-
cle size distribution. The absence of the fine particles
in the spent FCC is due to its loss from the unit with
the regenerator flue gas and the reactor vapour. The
bioleached FCC (Fig. 1c) again showed the absence
of fines, as well as evidence of small broken particles,
possibly due to the effect of bioleaching.
3.1.3. Toxicity characteristic leaching procedure
tests
Table 3 compares the TCLP tests results of spent
and bioleached catalysts with the TCLP regulatory
level set by U.S. EPA (U.S. Environmental Protection
Agency, 1998a) and the treatment standards for haz-
ardous waste for spent hydrorefining catalysts set by
U.S. EPA (U.S. Environmental Protection Agency,
1998b),as wellasthe recommendedacceptancecriteria
for disposal set by the National Environment Agency,
Singapore (National Environment Agency, 2003). Al-
though there are currently no regulations on the dis-
posaland storageofFCC catalysts,Furimsky (1996)ar-
guedthatit is reasonable to assume that most ofthereg-
ulations applicable to spent hydroprocessing catalysts
can be also applied for spent FCC catalysts. The TCLP
testsresults showedthat theconcentration of vanadium
and antimony in spent FCC exceeds the limit for the
treatment standards for hazardous waste for spent hy-
drorefining catalysts. It may be argued that spent FCC
catalyst exhibits toxicity characteristics and should be
treated before disposal. Although they are currently
classified as non-hazardous waste by the Environmen-
tal Protection Agency in the USA, it is certainly likely
that the disposal of spent FCC catalyst will be regu-
lated in the future. It is noteworthy that (Table 3) also
Fig. 1. SEM photomicrograph of FCC catalysts (120×magnifica-
tion): (a) fresh catalyst, (b) spent catalyst and (c) bioleached spent
catalyst.
showed that the concentration of metals was reduced
to well below the regulatory limits after bioleaching.
Thus, the bioleached spent catalyst may be disposed of
safely or reused in other processes.

K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170 165
Table 3
TCLP test results for spent (SFCC) and bioleached spent FCC (BSFCC) catalysts
Element Metal concentration in extraction fluid (mgl−1)
SFCC BSFCCaRegulatory levels (National
Environment Agency)bRegulatory levels (U.S.
EPA)cTreatment standards for
hazardous wastes (U.S. EPA)d
Al 1.45 ±0.07 1.02 ±0.00 ns ns ns
Ni 0.33 ±0.02 0.22 ±0.02 5 ns 11
V 5.62 ±0.15 0.37 ±0.01 ns ns 1.6
Sb 1.41 ±0.10 0.13 ±0.02 ns ns 1.15
Fe 0.05 ±0.01 0.03 ±0.04 100 ns ns
ns: not stated.
aBSFCC at 4% pulp density, two-step bioleaching, after 41 days.
bRecommendedacceptance criteria forsuitability ofindustrial wastes forlandfill disposalset by theNational EnvironmentAgency,Singapore.
c“Identification and listing of hazardous waste” U.S. Code of Federal Regulations (CFR), Title 40, Chapter 1, Part 261.
d“Treatment standards for hazardous waste” for spent hydrorefining catalysts. U.S. Code of Federal Regulations (CFR), Title 40, Chapter 1,
Part 261, Subpart D.
3.2. Bioleaching with A. niger
3.2.1. Determination of pre-incubation period in
two-step and spent medium leaching
Prior to the bioleaching processes, a pure culture
of A. niger was incubated until it reached the station-
ary phase in order to determine the optimum period
for addition of catalyst, as well as to obtain the spent
medium by filtration of the culture. Pure culture (with-
out spent catalyst) experiments were carried out under
identical conditions of bioleaching (see Section 2.5).
Fig. 2a shows the changes in concentration of sugars
andbiomassduring43 days of incubation. Sucrose was
completely hydrolyzed to glucose and fructose within
2 days of incubation, a result consistent with Hossain
et al. (1984). The glucose concentration was 4.5g l−1
at day 0 and dramatically increased to 38gl−1at day
2, beyond which a decrease was observed before it was
depletedafter 35days. Thedecreasein sugarconcentra-
tions was accompanied an increase in fungal biomass;
the biomass attained a constant dry weight of about
25gl−1during the stationary phase. Similar biomass
dryweight (26 g l−1) was alsoreportedby Santhiya and
Ting (2005).
Along with the sugar consumption, the primary
metabolites citric acid, oxalic acid and gluconic acid
were produced, with citric acid reaching a maximum
yieldofabout71mMattheendofincubation(Fig.2b).
pH decreased to 3.1 after 2 days incubation and re-
mained constant at about 2.7.
Theincrease in acid and biomass concentration, and
the complete hydrolysis of sucrose at the second day
Fig.2. (a)Concentration ofsugarsand biomass and(b) concentration
of organic acids and pH in pure culture of Aspergillus niger.
of incubation indicated that A. niger was in the active
growth phase. Thus, the spent catalyst was added to
the culture for bioleaching after 2 days of incubation
(under the two-step process). As 14 days of incubation

166 K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170
marked the beginning of the stationary phase, the spent
medium was obtained by filtering the culture after this
period.
3.2.2. Two-step bioleaching
A comparison between metal leaching efficiency
of one-step and two-step bioleaching process at
1% (w/v) pulp density showed that metal leach-
ing efficiency in the two-step process was higher
than that of one-step process (Fig. 3). Therefore,
two-step process was used for further bioleaching
studies.
Fig. 4a shows the changes in the sugar and biomass
concentrations during the two-step bioleaching at a 1%
(w/v) pulp density. The sucrose was fully hydrolysed
by A. niger before the addition of catalyst to the cul-
ture medium. The fungal biomass increased along with
a simultaneous decrease in the glucose and fructose
concentrations. Similar to the one-step process (results
Fig. 3. Metal leaching efficiency in one-step and two-step processes
at 1% (w/v) pulp density.
not shown), a faster consumption of glucose occurred
compared to that of fructose.
Fig. 4b shows the pH profile and acid production
during bioleaching. pH increased from an initial 3.12
Fig. 4. (a) Concentration of sugars and biomass, (b) concentration of organic acids and pH and (c) metal leaching efficiency in two-step
bioleaching at 1% (w/v) pulp density.

K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170 167
to 3.74 after the addition of catalyst due to its slightly
alkaline nature. Subsequently, pH dropped steadily to
2.8 after 28 days and gradually increased again. The
decrease in pH during fungal growth was due to the
productionof organic acids(citric,oxalic and gluconic)
(Bosshard et al., 1996; Castro et al., 2000; Brandl et al.,
2001). The concentration of citrate increased with time
and reached a maximum of 70mM after 32 days and
thereafter decreased and was no longer detectable af-
ter 51 days. (In pure culture and in one-step bioleach-
ing, the highest concentration of citrate was 71 and
50mM, respectively.) Oxalate secretion by the fungus
increased with time, and reached about 49mM after
51 days.
It was observed that the increase in the leaching of
the heavy metals paralleled the increase in the concen-
tration of organic acids (mainly citric acid; see Fig. 4b
and c). This phenomenon indicated that the biogeni-
cally produced organic acids played a direct and im-
portant role in the bioleaching process. The metal ex-
traction efficiency for antimony was the highest at 64%
after 32 days. The highest metal leaching efficiencies
were reached after 46 days for all other metals (Al,
V, Fe and Ni). The decrease in leaching efficiency be-
yond the maximum is probably due to precipitation of
unknown insoluble products.
Fig. 5 shows the metal leaching efficiencies of the
two-step process at various pulp densities. The opti-
mum pulp density for bioleaching was 1% (w/v) for
almost all the metals investigated as was the case in
Santhiya and Ting (2005). The decrease in the metal
Fig. 5. Metal leaching efficiency at various pulp densities in a two-
step process.
leaching efficiency with an increase in the pulp den-
sity is mainly due to the inability of the fungus to
grow well under high concentration of heavy metals
in the spent catalyst at higher pulp densities. Since bi-
oleaching of heavy metals by the fungus is mainly de-
pendent on the metabolites produced (Burgstaller and
Schinner, 1993; Gadd and Sayer, 2000), the lower
leaching efficiency observed at a higher pulp density
is to be expected.
3.2.3. Spent medium leaching
Spent medium leaching at various pulp densities (1,
2, 4 and 8%) was also conducted. The main leaching
agent in the spent medium was citric acid, which was
produced at approximately 57mM after 14 days (See
Fig. 2b). The concentration of gluconic acid and oxalic
acid were 47.7 and 7.2mM, respectively. The amount
of organic acids produced depends on many factors
including the buffering capacity of medium, the carbon
source, the ratio of nitrogen and phosphate in medium,
and the experimental conditions for the fungal growth
(Burgstaller and Schinner, 1993).
The pH of the spent medium harvested after 14
days incubation of the culture was 2.72, but increased
marginally with the addition of spent catalysts. A
higher pulp density resulted in a higher pH, since the
catalystwasslightly alkaline. pH remained almost con-
stantthroughout the leaching periodsinceno additional
organic acid was produced. As expected, the metal
leaching efficiency of spent medium decreased as pulp
density increased (Fig. 6).
Fig. 6. Metal leaching efficiency of spent medium at various pulp
densities.

168 K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170
Fig. 7. Metal leaching efficiency in various leaching processes at 2%
(w/v) pulp density.
3.2.4. Metal leaching efficiency under various
leaching processes
Themetalleachingefficienciesunder various leach-
ing processes (at 2% (w/v) pulp density) are shown in
Fig. 7. Bioleaching consistently resulted in the highest
metal leaching efficiency for all metals investigated.
This is followed by spent medium leaching which
generally attained about 63–93% efficiency achiev-
able with bioleaching. The controls (i.e. fresh sucrose
medium and deionised water) were able to leach only
vanadium, antimony and nickel, and with <17% leach-
ing efficiency.
It has been proposed that the higher leaching yield
in bioleaching compared to spent medium leaching
may due to bioaccumulation occurring in the for-
mer (Burgstaller and Schinner, 1993; Sayer and Gadd,
1997). Bosshard et al. (1996) also claimed bioaccumu-
lation as one of the mechanisms of fungal bioleach-
ing, and reported an approximately 5% higher leach-
ing efficiency in bioleaching than spent medium for
Al, Fe, Mn and Ni. Castro et al. (2000) also reported
higher concentrations of zinc and nickel in bioleach-
ing by A. niger compared to using the supernatant
liquor.
Although spent medium generally gave lower metal
leaching efficiency than bioleaching processes, it has
the advantages of easier handling and a shorter pro-
cessing time. Spent medium leaching also allows for
optimization (Bosshard et al., 1996), since the produc-
tion of the organic acids and the leaching process may
be decoupled and optimized separately, and hence the
Fig. 8. Metal leaching efficiency of various organic acids (100mM)
at 2% (w/v) pulp density.
issue of metal toxicity to the fungi at higher pulp den-
sity no longer arises.
3.2.5. Mass balances
A mass balance of the metals in the spent catalyst
was performed by summing the concentration of the
leached metals in the medium and the residual metals
in the solids associated with the biomass and the spent
catalysts. Results showed that except for Al (22%) and
Fe (12%), significantly less than 10% difference was
obtained for Fe, Ni, Sb and V.
3.3. Comparison between chemical leaching and
bioleaching
Bosshard et al. (1996) have shown that acid concen-
tration has a pronounced effect on leaching efficiency;
the leaching efficiencies for all the metals tested was
reduced to less than 2% when the citric acid concen-
tration was decreased 10 times. In our study, the leach-
ing efficiency of the metals increased with increasing
concentration of the acids (results not shown). Fig. 8
comparesthemetalleachingefficiencyof various acids
at 100mM at 2% (w/v) pulp density after 3 days incu-
bation. Oxalic acid showed the highest leaching effi-
ciency while gluconic acid showed the lowest leaching
efficiency. The latter is likely to be due to the higher pH
of gluconic acid; the average pH of gluconic acid (at
20, 50 and 100mM) was six whereas that of the other
acids was less than two.

K.M.M. Aung, Y.-P. Ting / Journal of Biotechnology 116 (2005) 159–170 169
Fig.9. Metal leaching efficiency inchemical and biological leaching
at 2% (w/v) pulp density.
A comparison was made between chemical leach-
ing and bioleaching, using a mixture of commercial
citric, gluconic and oxalic acids at the same concen-
tration of the organic acids produced in the two-step
bioleachingprocessat2%(w/v) pulp density.The mix-
turecontained 85.5 mMcitrate,13.6 mM gluconateand
no oxalate. Spent catalyst at 2% (w/v) pulp density was
used for both leaching. Leaching was terminated when
the metal concentrations in the leachate were relatively
constant.
Generally, bioleaching gave 2.7–20% higher metal
extraction efficiency than chemical leaching with com-
mercialorganicacidsat thesame concentration(Fig.9).
These results corroborate the work of Illmer et al.
(1995), who reported that metal leaching efficiency of
chemical leaching using a mixture of 30mM citrate,
30 mM of gluconate and 1mM of oxalic was distinctly
below the efficiency of biotic leaching even though
acids were employed in higher concentrations than
observed in culture solution of A. niger.Kamali and
Mulligan (2002) also reported that chemical leaching
efficiencyof copperfromlow-grade ores bycitric(25%
extraction) and oxalic acids (7% extraction) was lower
than the biotic leaching by A. niger (60% extraction).
Castro et al. (2000) also showed that chemical leaching
using citric acid was less effective in the extraction of
nickel from garnierite compared to bioleaching by A.
niger. These results affirmed the statement by Schinner
and Burgstaller (1989) who reported that secondary
metabolites might also add to the process of leaching.
4. Conclusion
This work has shown that heavy metals from spent
industrial catalyst may be mobilised by leaching with
A. niger. The extraction of metals was generally higher
atlower pulpdensities;the highest extractionefficiency
was achieved with 1% (w/v) catalyst. Biological leach-
ing was more effective than chemical leaching. The
more favourable results obtained in the bioleaching
process suggest that the mechanism is not simply a
direct chemical attack on the catalyst but that the fun-
gus participates in the leaching process. Bioleaching
also gave rise to higher metal extractions than the fresh
medium and the cell-free spent medium. pH decreased
with time during bioleaching, but remained relatively
constant in both the fresh medium and the cell-free
spent medium leaching, thus indicating that the mi-
croorganisms play a role in effecting metal extraction
from the spent catalyst.
Acknowledgements
The authors would like to thank Dr. H. Brandl for
the supply of the Aspergillus niger used in this work.
Thiswork was supported under the NationalUniversity
of Singapore research grant RP 279-000-059-112.
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